The control of gene expression by the large class of small (~22nt), noncoding microRNAs has emerged as a new mode of post-transcriptional gene regulation in animal development and disease. The microRNA-induced silencing complex (miRISC) guides microRNAs to the 3< UTR of target mRNAs through partial base pairing and leads to translational inhibition and/or target degradation. However, the steps of target recognition and binding, and the role of miRISC and other RNA-associated factors remain active areas of investigation. In this proposal we will determine the function of a novel protein complex that binds microRNAs and their targets in the nematode, C. elegans. This complex includes AIN-1, a conserved member of miRISC, as well as three highly conserved RNA binding proteins with Cold Shock Domains (CSD). We hypothesize that the CSD proteins function as key factors for repressing the expression of microRNA targets. To elucidate the function of the CSD proteins, we will focus our studies on the regulation of miR-1 mRNA targets in the body muscle of C. elegans. Our previous work on miR-1 and its regulation of synaptic signaling at the neuromuscular junction establishes the experiment framework for our current proposal. We will test the requirement for the CSD proteins in miR-1 target expression, binding to the AIN-1 complex, and recruitment to P bodies. We will test our hypothesis in the following two aims: In Aim 1, we will investigate the function of the CSD proteins in the regulation of miR- 1 target expression and binding to the AIN-1 complex. We hypothesize that the CSD proteins recruit the miR-1 target mRNAs, directly or indirectly, to the AIN-1 complex for subsequent target repression. In Aim 2, we will test the role of the CSD proteins in AIN-1 complex formation and recruitment to the P bodies, the cytoplasmic centers for repression of microRNA target mRNAs. We hypothesize that binding of mRNA substrates by the CSD proteins is a required step for AIN-1 complex formation and targeting to P bodies. Finally, we will test the hypothesis of whether individual CSD proteins form distinct, modular AIN-1 complexes, perhaps as a way to regulate subsets of microRNA targets. We expect that these studies will provide insights into the conserved mechanisms of how microRNA targets are recognized and designated for translational inhibition or degradation.